RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4073
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0910900; Gene model (P.falciparum): PF3D7_1137800; Gene product: sporozoite surface protein essential for liver stage development, putative (SPELD)
Name tag: mCherry
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 14 January 2017, 20:39
  *RMgm-4073
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28067322
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherAl-Nihmi FM, Kumar KA
Name Group/DepartmentDepartment of Animal Biology, School of Life Sciences
Name InstituteUniversity of Hyderabad
CityHyderabad
CountryIndia
Name of the mutant parasite
RMgm numberRMgm-4073
Principal namepbspeldmCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystAt day 14, midgut oocysts, sporozoites within oocyst and at day 18–21, the salivary glands sporozoites were all positive for mCherry expression. Deconvoluted images of intact salivary glands harbouring sporozoites revealed the localization of mCherry to the sporozoite plasma membrane. The membrane associated mCherry colocalised with CSP in both midgut and salivary gland sporozoites.
SporozoiteAt day 14, midgut oocysts, sporozoites within oocyst and at day 18–21, the salivary glands sporozoites were all positive for mCherry expression. Deconvoluted images of intact salivary glands harbouring sporozoites revealed the localization of mCherry to the sporozoite plasma membrane. The membrane associated mCherry colocalised with CSP in both midgut and salivary gland sporozoites.
Liver stageAt day 14, midgut oocysts, sporozoites within oocyst and at day 18–21, the salivary glands sporozoites were all positive for mCherry expression. Deconvoluted images of intact salivary glands harbouring sporozoites revealed the localization of mCherry to the sporozoite plasma membrane. The membrane associated mCherry colocalised with CSP in both midgut and salivary gland sporozoites. The localization of mCherry was observed to the membrane of developing EEF at 17 hrs that colocalised with UIS4, a parasitophorous vacuolar membrane protein. However, no mcherry expression was associated with EEFs at 26, 36 and 62 hours post infection.
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal tagged mCherry version of SPELD

Protein (function)
SPELD was identified as the most abundantly expressed sequence tag in a sporozoite SAGE transcriptome analysis. Homologs of SPELD were not detected in other apicomplexa. PbSPELD sequence was analysed for the presence of transmembrane regions using multiple programs (TMHMM, TMpred, DAS, TopPred2), which predicted a single transmembrane region that is shared by all Plasmodium SPELD proteins. The SPELD proteins contain a tyrosine-rich domain, and the most abundant amino acid is tyrosine.
Phenotype analyses of a mutant lacking expression of SPELD (RMgm-4072) showed strongly reduced infectivity of sporozoites that was the result of aberrant EEF development

Phenotype
No expression in asexual stages,gametocytes, ookinetes and young oocysts.
At day 14, midgut oocysts, sporozoites within oocyst and at day 18–21, the salivary glands sporozoites were all positive for mCherry expression. Deconvoluted images of intact salivary glands harbouring sporozoites revealed the localization of mCherry to the sporozoite plasma membrane. The membrane associated mCherry colocalised with CSP in both midgut and salivary gland sporozoites. The localization of mCherry was observed to the membrane of developing EEF at 17 hrs that colocalised with UIS4, a parasitophorous vacuolar membrane protein. However, no mcherry expression was associated with EEFs at 26, 36 and 62 hours post infection.

Additional information

Other mutants
A mutant lacking expression of SPELD (RMgm-4072)


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0910900
Gene Model P. falciparum ortholog PF3D7_1137800
Gene productsporozoite surface protein essential for liver stage development, putative
Gene product: Alternative nameSPELD
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6